The proposed research is to define the signaling mechanisms important in physiology and virulence of the basidiomyceteous Cryptococcus neoformans. This organism is the leading cause of fungal meningitis in immunocompromised individuals and a haploid pathogen with a well-defined life cycle that is amenable to genetic and molecular analysis, making it an important and attractive model to study cellular growth and virulence. The rationale for this proposal is further strengthened by both our previous studies, and those of others, on GTP-binding signaling proteins that have resulted in the identification of several key cryptococcal genes such as GPA1 and GPB1. Gpa1 is a G-protein alpha subunit that governs a signaling pathway sensing glucose and attenuating virulence (1, 2) and Gpb1 is a G-protein beta subunit that mediates mating via a conserved MAP kinase cascade (3, 4). The proposed study focuses on how G-protein a signaling is regulated. I have identified the CRG1 (Cryptococcal Regulator of G-protein signaling) gene that encodes a homolog of the RGS protein family in C. neoformans and have shown that Crg1 functions in mating and virulence. Functional characterization of Crg1 provides a unique vantage point from which to elucidate the mechanisms of G-protein signaling important for fungal growth, differentiation, and pathogenesis. I will test the hypothesis that Crg1 exhibits an RGS function by accelerating the hydrolysis of GTP-Galpha to GDP-Galpha to negatively modulate G-protein signaling, and thereby plays a pivotal role in growth and pathogenesis. Crg1 mutant strains will be examined in two divergent but related serotype A and D strains that represent distinct varieties of C. neoformans. Interactions between Crg1 and Galpha proteins will be examined. In addition, the role of Crg1 in virulence will be evaluated. Finally, a surrogate host will be used to identify additional regulatory proteins that also govern G-protein signaling. The long-term goal of this investigation is to understand how C. neoformans cells sense and respond to signals that coordinate their growth and differentiation, to reveal the relationships between signaling pathways and pathogenesis, and to discover novel antifungal targets for the treatment of cryptococcal infection.